Oligomer-protein tyrosine kinase inhibitor conjugates

ABSTRACT

The invention relates to (among other things) oligomer-PTK inhibitor conjugates and related compounds. A compound of the invention, when administered by any of a number of administration routes, exhibits advantages over PTK inhibitor compounds lacking a water-soluble, non-peptidic oligomer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/963,562, filed 9 Aug. 2013, now U.S. Pat. No.8,921,371, which is a continuation application of U.S. patentapplication Ser. No. 13/264,519, filed 7 Dec. 2011, now U.S. Pat. No.8,530,492, which is a 35 U.S.C. §371 application of InternationalApplication No. PCT/US2010/001162, filed 19 Apr. 2010, designating theUnited States, which claims the benefit of priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application Ser. No. 61/170,541,filed 17 Apr. 2009, and U.S. Provisional Patent Application Ser. No.61/267,302, filed 07 Dec. 2009, the disclosures of which areincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention comprises (among other things) chemically modifiedprotein tyrosine kinase inhibitors (PTK inhibitors) that possess certainadvantages over PTK inhibitors lacking the chemical modification. Thechemically modified PTK inhibitors described herein relate to and/orhave application(s) in (among others) the fields of drug discovery,pharmacotherapy, physiology, organic chemistry and polymer chemistry.

BACKGROUND OF THE INVENTION

Protein kinases (“PKs”) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues of proteins.The consequences of this seemingly simple activity are staggering; cellgrowth, differentiation and proliferation, i.e., virtually all aspectsof cell life in one way or another depend on PK activity. Furthermore,abnormal PK activity has been related to a host of disorders, rangingfrom relatively non-life threatening diseases such as psoriasis toextremely virulent diseases such as glioblastoma (brain cancer).

Generally, the PKs can be categorized into two classes, the proteintyrosine kinases (PTKs) and the serine-threonine kinases (STKs).However, other kinases are reported that phosphorylate other aminoacids, such as histidine. Kinases with dual (serine/threonine andtyrosine) specificity are also reported (e.g., MEK or MAPKK).

Many PTKs are involved with growth factor receptors. When bound by agrowth factor ligand, growth factor receptors are converted to an activeform which interacts with proteins on the inner surface of the cellmembrane. This leads to phosphorylation on tyrosine residues of thereceptor and other proteins and to the formation inside the cell ofcomplexes with a variety of cytoplasmic signaling molecules that, inturn, affect numerous cellular responses such as cell division(proliferation), cell differentiation, and cell growth.

Growth factor receptors with PTK activity are known as receptor tyrosinekinases (“RTKs”). They comprise a family of transmembrane receptors withdiverse biological activity. The HER subfamily of RTKs includes EGFR(epithelial growth factor receptor), HER2, HER3 and HER4. These RTKsconsist of an extracellular glycosylated ligand binding domain, atransmembrane domain and an intracellular cytoplasmic catalytic domainthat can phosphorylate tyrosine residues on proteins.

Another RTK subfamily consists of insulin receptor (IR), insulin-likegrowth factor I receptor (IGF-1R) and insulin receptor related receptor(IRR). IR and IGF-1R interact with insulin, IGF-I and IGF-II to form aheterotetramer of two entirely extracellular glycosylated alpha subunitsand two beta subunits which cross the cell membrane and which containthe tyrosine kinase domain.

A third RTK subfamily is referred to as the “platelet derived growthfactor receptor” (“PDGFR”) group, which includes PDGFR-α, PDGFR-β,CSFIR, c-kit and c-fms. These receptors consist of glycosylatedextracellular domains composed of variable numbers of immunoglobin-likeloops and an intracellular domain wherein the tyrosine kinase domain isinterrupted by unrelated amino acid sequences.

Another group, which, because of its similarity to the PDGFR subfamily(and is sometimes subsumed into the PDGFR subfamily) is the fetus liverkinase (“flk”) receptor subfamily. This group is believed to be made upof kinase insert domain-receptor fetal liver kinase-1 (KDR/FLK-1,VEGF-R2), flk-1R, flk-4 and fins-like tyrosine kinase 1 (flt-1).

A further member of the tyrosine kinase growth factor receptor family isthe fibroblast growth factor (“FGF”) receptor subgroup. This groupconsists of four receptors, FGFR1-4, and seven ligands, FGF1-7. Whilenot yet well defined, it appears that the receptors consist of aglycosylated extracellular domain containing a variable number ofimmunoglobin-like loops and an intracellular domain in which thetyrosine kinase sequence is interrupted by regions of unrelated aminoacid sequences.

Still another member of the tyrosine kinase growth factor receptorfamily is the vascular endothelial growth factor (“VEGF”) receptorsubgroup. VEGF is a dimeric glycoprotein similar to PDGF but hasdifferent biological functions and target cell specificity in vivo. Inparticular, VEGF is presently thought to play an essential role isvasculogenesis and angiogenesis.

In addition to the RTKs, there also exists a family of entirelyintracellular PTKs called “non-receptor tyrosine kinases” or “cellulartyrosine kinases” (CTKs). CTKs do not contain extracellular andtransmembrane domains. More than 24 CTKs in 11 subfamilies (Src, Frk,Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified.The Src subfamily appears so far to be the largest group of CTKs andincludes Src, Yes, Fyn, Lyn, Lek, Bik, Hck, Fgr and Yrk.

The serine/threonine kinases, STKs, like the CTKs, are predominantlyintracellular although there are a few receptor kinases of the STK type.STKs are the most common of the cytosolic kinases.

RTKs, CTKs and STKs have all been implicated in a host of pathogenicconditions including, significantly, various cancers. Other pathogenicconditions which have been associated with PTKs include, withoutlimitation, psoriasis, hepatic cirrhosis, diabetes, angiogenesis,restenosis, ocular diseases, rheumatoid arthritis and other inflammatorydisorders, immunological disorders such as autoimmune disease,cardiovascular disease such as atherosclerosis and a variety of renaldisorders.

With regard to cancer, two of the major hypotheses advanced to explainthe excessive cellular proliferation that drives tumor developmentrelate to functions known to be PK regulated. It has been suggested thatmalignant cell growth results from a breakdown in the mechanisms thatcontrol cell division and/or differentiation. It has been shown that theprotein products of a number of proto-oncogenes are involved in thesignal transduction pathways that regulate cell growth anddifferentiation. These protein products of proto-oncogenes include theextracellular growth factors, transmembrane growth factor PTK receptors(RTKs), cytoplasmic PTKs (CTKs) and cytosolic STKs, discussed above.

Many small molecule PTK inhibitors have been developed over the years,including, but not limited to, imatinib, dasatinib, canertinib,erlotinib, gefitinib, lapatinib, sorafenib, sunitinib, and vatalinib.These molecules have been prescribed for many diseases, including,chronic myelogenous leukemia (CML), gastrointestinal stromal tumors(GISTs), renal cell carcinoma, and solid tumors, including breast, lung,and colorectal cancers; and are used as anti-neoplastic agents and asradio-sensitizing agents. However, treatment with these agents sufferfrom many side effects, including, hypertension, fatigue, asthenia,diarrhea, hand-foot syndrome, neutropenia and myelosuppression,peripheral edema, headache, and hypocalcemia.

Therefore, pharmacotherapy with such therapeutic PTK inhibitors would beimproved if these and/or other adverse or side effects associated withtheir use could be decreased or if their pharmacology may be improved.Thus, there is a large unmet need for developing novel PTK inhibitorcompounds.

The present invention seeks to address these and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a compound is provided, thecompound comprising a PTK inhibitor residue covalently attached via astable or degradable linkage to a water-soluble, non-peptidic oligomer.

The “PTK inhibitor residue” is a compound having a structure of a PTKinhibitor compound that is altered by the presence of one or more bonds,which bonds serve to attach (either directly or indirectly) one or morewater-soluble, non-peptidic oligomers.

Exemplary compounds of the invention include those having the followingstructure

wherein:

R₁ is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-loweralkyl-substituted phenyl wherein the amino group in each case is free,alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom, or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen;

R₂ and R₃ are each independently of the other hydrogen or lower alkyl;

one or two of the radicals R₄, R₅, R₆, R₇ and R₈ are each nitro,fluoro-substituted lower alkoxy or a radical of Formula II-C,—N(R₉)—C(═Z)—(Y)_(n)—R₁₀—X-POLY  (Formula II-C),wherein

R₉ is hydrogen or lower alkyl,

Z is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-loweralkyl-hydroximino,

Y is oxygen or —NH—,

n is 0 or 1,

R₁₀ is a bivalent aliphatic hydrocarbon radical having 5-22 carbonatoms, a phenyl or naphthyl radical, each of which is unsubstituted orsubstituted by a moiety selected from the group consisting of cyano,lower alkyl, hydroxy-lower alkyl, amino-lower alkyl,(4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-loweralkylamino, lower alkanoylamino, benzoylamino, carboxy, loweralkoxycarbonyl, phenyl-lower alkyl wherein the phenyl radical isunsubstituted or substituted, a cycloalkyl or cycloalkenyl radicalhaving up to 30 carbon atoms, cycloalkyl-lower alkyl orcycloalkenyl-lower alkyl each having up to 30 carbon atoms in thecycloalkyl or cycloalkenyl moiety, a monocyclic radical having 5 or 6ring members and 1-3 ring hetero atoms selected from nitrogen, oxygenand sulfur, to which radical one or two benzene radicals may be fused,and lower alkyl substituted by a monocyclic radical,

X is a spacer moiety, and

POLY is a water-soluble, non-peptidic oligomer; and

the remaining radicals of R₄, R₅, R₆, R₇ and R₈ that are not nitro,fluoro-substituted lower alkoxy or a radical of formula II are eachindependently selected from the group consisting of hydrogen, loweralkyl that is unsubstituted or substituted by free or alkylated amino,piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or loweralkanoyl, trifluoromethyl, free, etherified or esterifed hydroxy, free,alkylated or acylated amino or free or esterified carboxy.

Exemplary compounds of the invention include those moieties of FormulaI-C wherein: R₁ is unsubstituted or C₁₋₇ alkyl-substituted pyridylbonded at a ring carbon atom; R₂ is selected from the group consistingof H, methyl and ethyl; R₃ is selected from the group consisting of H,methyl and ethyl; only one of R₄, R₅, R₆, R₇ and R₈ is a radical of—N(R₉)—C(═Z)—(Y)_(n)—R₁₀—X-POLY (Formula II-C), and the remaining of R₄,R₅, R₆, R₇ and R₈ are each independent selected from the group ofhydrogen, methyl and ethyl, and further wherein, with respect to FormulaII-C, R₉ is selected from the group consisting of H, methyl and ethyl, Zis oxo, (n) is 0, and R₁₀ is a bivalent aromatic radical (such as2-naphthyl and phenyl) optionally substituted with cyano, hydroxyl,amino, and 4-methyl-piperazinyl substituted methyl, X is a spacermoiety, and POLY is a water-soluble, non-peptidic oligomer.

In this regard, any PTK inhibitor compound having PTK inhibitoryactivity can be used as a PTK inhibitor moiety. Exemplary PTK inhibitormoieties have a structure encompassed by Formula I:

wherein:

R₁ is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-loweralkyl-substituted phenyl wherein the amino group in each case is free,alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom, or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen;

R₂ and R₃ are each independently of the other hydrogen or lower alkyl;

one or two of the radicals R₄, R₅, R₆, R₇ and R₈ are each nitro,fluoro-substituted lower alkoxy or a radical of Formula II,—N(R₉)—C(═Z)—(Y)_(n)—R₁₀  (Formula II),wherein

R₉ is hydrogen or lower alkyl,

Z is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-loweralkyl-hydroximino,

Y is oxygen or —NH—,

n is 0 or 1,

R₁₀ is an aliphatic hydrocarbon radical having 5-22 carbon atoms, aphenyl or naphthyl radical, each of which is unsubstituted orsubstituted by a moiety selected from the group consisting of cyano,lower alkyl, hydroxy-lower alkyl, amino-lower alkyl,(4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-loweralkylamino, lower alkanoylamino, benzoylamino, carboxy, loweralkoxycarbonyl, phenyl-lower alkyl wherein the phenyl radical isunsubstituted or substituted, a cycloalkyl or cycloalkenyl radicalhaving up to 30 carbon atoms, cycloalkyl-lower alkyl orcycloalkenyl-lower alkyl each having up to 30 carbon atoms in thecycloalkyl or cycloalkenyl moiety, a monocyclic radical having 5 or 6ring members and 1-3 ring hetero atoms selected from nitrogen, oxygenand sulfur, to which radical one or two benzene radicals may be fused,and lower alkyl substituted by a monocyclic radical; and

the remaining radicals of R₄, R₅, R₆, R₇ and R₈ that are not nitro,fluoro-substituted lower alkoxy or a radical of formula II are eachindependently selected from the group consisting of hydrogen, loweralkyl that is unsubstituted or substituted by free or alkylated amino,piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or loweralkanoyl, trifluoromethyl, free, etherified or esterifed hydroxy, free,allcylated or acylated amino or free or esterified carboxy.

Exemplary PTK inhibitor moieties include those moieties of Formula Iwherein: R₁ is unsubstituted or C₁₋₇ alkyl-substituted pyridyl bonded ata ring carbon atom; R₂ is selected from the group consisting of H,methyl and ethyl; R₃ is selected from the group consisting of H, methyland ethyl; only one of R₄, R₅, R₆, R₇ and R₈ is a radical of—N(R₉)—C(═Z)—(Y)_(n)—R₁₀ (Formula II), and the remaining of R₄, R₅, R₆,R₇ and R₈ are each independent selected from the group of hydrogen,methyl and ethyl, and further wherein, with respect to Formula II, R₉ isselected from the group consisting of H, methyl and ethyl, Z is oxo, (n)is 0, and R₁₀ is an aromatic radical (such as 2-naphthyl and phenyl)optionally substituted with cyano, hydroxyl, amino, and4-methyl-piperazinyl substituted methyl.

A further exemplary PTK inhibitor moiety is referred to generically asimatinib and chemically as(4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide),as well as its pharmaceutically acceptable salts including, but notlimited to, the mesylate salt (e.g., formed using methanesulfonic acid).

In one or more embodiments of the invention, a composition is provided,the composition comprising a compound comprising a PTK inhibitor residuecovalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer, and optionally, a pharmaceuticallyacceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a PTK inhibitor residuecovalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer, wherein the compound is present ina dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to a PTK inhibitor moiety.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound to a mammal in need thereof,comprising a PTK inhibitor residue covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer.

Additional embodiments of the presently described compounds,compositions, methods, and the like will be apparent from the followingdescription, examples, and claims. As can be appreciated from theforegoing and following description, each and every feature describedherein, and each and every combination of two or more of such features,is included within the scope of the present disclosure provided that thefeatures included in such a combination are not mutually inconsistent.In addition, any feature or combination of features may be specificallyexcluded from any embodiment of the present invention. Additionalaspects and advantages of the present invention are set forth in thefollowing description and claims, particularly when considered inconjunction with the accompanying examples and drawings.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of PTK inhibitorresidues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer comprises one or more monomersserially attached to form a chain of monomers. The oligomer can beformed from a single monomer type (i.e., is homo-oligomeric) or two orthree monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 1 to about 30monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though, the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG oligomers for use in thepresent invention will comprise one of the two following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG oligomers, thevariable (n) ranges from about 1 to 30, and the terminal groups andarchitecture of the overall PEG can vary. When PEG further comprises afunctional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer does notresult in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxidelinkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike. In addition, the end-capping group may contain a targeting moiety.

The term “targeting moiety” is used herein to refer to a molecularstructure that helps the conjugates of the invention to localize to atargeting area, e.g., help enter a cell, or bind a receptor. Preferably,the targeting moiety comprises a vitamin, antibody, antigen, receptor,DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell-specificlectins, steroid or steroid derivative, RGD peptide, ligand for a cellsurface receptor, serum component, or combinatorial molecule directedagainst various intra- or extracellular receptors. The targeting moietymay also comprise a lipid or a phospholipid. Exemplary phospholipidsinclude, without limitation, phosphatidylcholines, phospatidylserine,phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine.These lipids may be in the form of micelles or liposomes and the like.The targeting moiety may further comprise a detectable label oralternately a detectable label may serve as a targeting moiety. When theconjugate has a targeting group comprising a detectable label, theamount and/or distribution/location of the polymer and/or the moiety(e.g., active agent) to which the polymer is coupled can be determinedby using a suitable detector. Such labels include, without limitation,fluorescers, chemiluminescers, moieties used in enzyme labeling,colorimetric (e.g., dyes), metal ions, radioactive moieties, goldparticles, quantum dots, and the like.

“Branched,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymers “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(typically through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, N.Y., 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity. “Monodisperse” refers to anoligomer composition wherein substantially all of the oligomers in thecomposition have a well-defined, single molecular weight and definednumber of monomers, as determined by chromatography or massspectrometry. Monodisperse oligomer compositions are in one sense pure,that is, substantially having a single and definable number (as a wholenumber) of monomers rather than a large distribution. A monodisperseoligomer composition possesses a MW/Mn value of 1.0005 or less, and morepreferably, a MW/Mn value of 1.0000. By extension, a compositioncomprised of monodisperse conjugates means that substantially alloligomers of all conjugates in the composition have a single anddefinable number (as a whole number) of monomers rather than a largedistribution and would possess a MW/Mn value of 1.0005, and morepreferably, a MW/Mn value of 1.0000 if the oligomer were not attached tothe therapeutic moiety. A composition comprised of monodisperseconjugates may, however, include one or more nonconjugate substancessuch as solvents, reagents, excipients, and so forth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is generally symmetric about its mean, although the size of the twopeaks may differ. Ideally, the polydispersity index of each peak in thebimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 orless, and even more preferably 1.0005 or less, and most preferably aMW/Mn value of 1.0000. By extension, a composition comprised of bimodalconjugates means that substantially all oligomers of all conjugates inthe composition have one of two definable and different numbers (aswhole numbers) of monomers rather than a large distribution and wouldpossess a MW/Mn value of 1.01 or less, more preferably 1.001 or less andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000 if the oligomer were not attached to the therapeutic moiety. Acomposition comprised of bimodal conjugates may, however, include one ormore nonconjugate substances such as solvents, reagents, excipients, andso forth.

A “PTK inhibitor” is broadly used herein to refer to an organic,inorganic, or organometallic compound having a molecular weight of lessthan about 1000 Daltons and having some degree of activity as a PTKinhibitor. PTK inhibitor activity of a compound may be measured byassays known in the art and also as described herein.

A “biological membrane” is any membrane made of cells or tissues thatserves as a barrier to at least some foreign entities or otherwiseundesirable materials. As used herein a “biological membrane” includesthose membranes that are associated with physiological protectivebarriers including, for example: the blood-brain barrier (BBB); theblood-cerebrospinal fluid barrier; the blood-placental barrier; theblood-milk barrier; the blood-testes barrier; and mucosal barriersincluding the vaginal mucosa, urethral mucosa, anal mucosa, buccalmucosa, sublingual mucosa, and rectal mucosa. Unless the context clearlydictates otherwise, the term “biological membrane” does not includethose membranes associated with the middle gastro-intestinal tract(e.g., stomach and small intestines).

A “biological membrane crossing rate,” provides a measure of acompound's ability to cross a biological membrane, such as theblood-brain barrier (“BBB”). A variety of methods may be used to assesstransport of a molecule across any given biological membrane. Methods toassess the biological membrane crossing rate associated with any givenbiological barrier (e.g., the blood-cerebrospinal fluid barrier, theblood-placental barrier, the blood-milk barrier, the intestinal barrier,and so forth), are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart.

A “reduced rate of metabolism” refers to a measurable reduction in therate of metabolism of a water-soluble oligomer-small molecule drugconjugate as compared to the rate of metabolism of the small moleculedrug not attached to the water-soluble oligomer (i.e., the smallmolecule drug itself) or a reference standard material. In the specialcase of “reduced first pass rate of metabolism,” the same “reduced rateof metabolism” is required except that the small molecule drug (orreference standard material) and the corresponding conjugate areadministered orally. Orally administered drugs are absorbed from thegastro-intestinal tract into the portal circulation and may pass throughthe liver prior to reaching the systemic circulation. Because the liveris the primary site of drug metabolism or biotransformation, asubstantial amount of drug may be metabolized before it ever reaches thesystemic circulation. The degree of first pass metabolism, and thus, anyreduction thereof, may be measured by a number of different approaches.For instance, animal blood samples may be collected at timed intervalsand the plasma or serum analyzed by liquid chromatography/massspectrometry for metabolite levels. Other techniques for measuring a“reduced rate of metabolism” associated with the first pass metabolismand other metabolic processes are known, described herein and/or in therelevant literature, and/or may be determined by one of ordinary skillin the art. Preferably, a conjugate of the invention may provide areduced rate of metabolism reduction satisfying at least one of thefollowing values: at least about 30%; at least about 40%; at least about50%; at least about 60%; at least about 70%; at least about 80%; and atleast about 90%. A compound (such as a small molecule drug or conjugatethereof) that is “orally bioavailable” is one that preferably possessesa bioavailability when administered orally of greater than 25%, andpreferably greater than 70%, where a compound's bioavailability is thefraction of administered drug that reaches the systemic circulation inunmetabolized form.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced. An“alkenyl” group is an alkyl of 2 to 20 carbon atoms with at least onecarbon-carbon double bond.

The terms “substituted alkyl” or “substituted C_(q-r) alkyl” where q andr are integers identifying the range of carbon atoms contained in thealkyl group, denotes the above alkyl groups that are substituted by one,two or three halo (e.g., F, Cl, Br, I), trifluoromethyl, hydroxy, C₁₋₇alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and soforth), C₁₋₇ alkoxy, C₁₋₇ acyloxy, C₃₋₇ heterocyclic, amino, phenoxy,nitro, carboxy, acyl, cyano. The substituted alkyl groups may besubstituted once, twice or three times with the same or with differentsubstituents.

“Lower alkyl” refers to an alkyl group containing from 1 to 7 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl. “Lower alkenyl” refers to a loweralkyl group of 2 to 6 carbon atoms having at least one carbon-carbondouble bond.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy, etc.),preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention causes no significant adverse toxicological effects to apatient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthalenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g., 1-2, 1-3 or 1-4 substituents)chosen from halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

Chemical moieties are defined and referred to throughout primarily asunivalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless,such terms are also used to convey corresponding multivalent moietiesunder the appropriate structural circumstances clear to those skilled inthe art. For example, while an “alkyl” moiety generally refers to amonovalent radical (e.g., CH₃—CH₂—), in certain circumstances, abivalent linking moiety can be “alkyl,” in which case those skilled inthe art will understand the alkyl to be a divalent radical (e.g.,—CH₂—CH₂—), which is equivalent to the term “alkylene.” (Similarly, incircumstances in which a divalent moiety is required and is stated asbeing “aryl,” those skilled in the art will understand that the term“aryl” refers to the corresponding multivalent moiety, arylene). Allatoms are understood to have their normal number of valences for bondformation (i.e., 1 for H, 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6for S, depending on the oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount may depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and may readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterodifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein, and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a PTK inhibitor residue covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer.

The “PTK inhibitor residue” is a compound having a structure of a PTKinhibitor compound that is altered by the presence of one or more bonds,which bonds serve to attach (either directly or indirectly) one or morewater-soluble, non-peptidic oligomers. Exemplary PTK inhibitors have astructure encompassed by Formula I:

wherein:

R₁ is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-loweralkyl-substituted phenyl wherein the amino group in each case is free,alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom, or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen;

R₂ and R₃ are each independently of the other hydrogen or lower alkyl;

one or two of the radicals R₄, R₅, R₆, R₇ and R₈ are each nitro,fluoro-substituted lower alkoxy or a radical of Formula II,—N(R₉)—C(═Z)—(Y)_(n)—R₁₀  (Formula II),wherein

R₉ is hydrogen or lower alkyl,

Z is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-loweralkyl-hydroximino,

Y is oxygen or —NH—,

n is 0 or 1,

R₁₀ is an aliphatic hydrocarbon radical having 5-22 carbon atoms, aphenyl or naphthyl radical, each of which is unsubstituted orsubstituted by a moiety selected from the group consisting of cyano,lower alkyl, hydroxy-lower alkyl, amino-lower alkyl,(4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-loweralkylamino, lower alkanoylamino, benzoylamino, carboxy, loweralkoxycarbonyl, phenyl-lower alkyl wherein the phenyl radical isunsubstituted or substituted, a cycloalkyl or cycloalkenyl radicalhaving up to 30 carbon atoms, cycloalkyl-lower alkyl orcycloalkenyl-lower alkyl each having up to 30 carbon atoms in thecycloalkyl or cycloalkenyl moiety, a monocyclic radical having 5 or 6ring members and 1-3 ring hetero atoms selected from nitrogen, oxygenand sulfur, to which radical one or two benzene radicals may be fused,and lower alkyl substituted by a monocyclic radical; and

the remaining radicals of R₄, R₅, R₆, R₇ and R₈ that are not nitro,fluoro-substituted lower alkoxy or a radical of formula II are eachindependently selected from the group consisting of hydrogen, loweralkyl that is unsubstituted or substituted by free or alkylated amino,piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or loweralkanoyl, trifluoromethyl, free, etherified or esterifed hydroxy, free,alkylated or acylated amino or free or esterified carboxy.

Exemplary PTK inhibitor moieties include those moieties of Formula Iwherein: R₁ is unsubstituted or C₁₋₇ alkyl-substituted pyridyl bonded ata ring carbon atom; R₂ is selected from the group consisting of H,methyl and ethyl; R₃ is selected from the group consisting of H, methyland ethyl; only one of R₄, R₅, R₆, R₇ and R₈ is a radical of—N(R₉)—C(═Z)—(Y)_(n)—R₁₀ (Formula II), and the remaining of R₄, R₅, R₆,R₇ and R₈ are each independent selected from the group of hydrogen,methyl and ethyl, and further wherein, with respect to Formula II, R₉ isselected from the group consisting of H, methyl and ethyl, Z is oxo, (n)is 0, and R₁₀ is an aromatic radical (such as 2-naphthyl and phenyl)optionally substituted with cyano, hydroxyl, amino, and4-methyl-piperazinyl substituted methyl.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a PTK inhibitor residue covalently attached via astable or degradable linkage to a water-soluble, non-peptidic oligomer,wherein the PTK inhibitor is imatinib(4-[(4-methylpiperazin-1-ypmethyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]phenyl]benzamide),and pharmaceutically acceptable salts thereof including, but not limitedto, the mesylate salt.

In some instances, PTK inhibitors can be obtained from commercialsources. In addition, PTK inhibitors can be obtained through chemicalsynthesis. Examples of PTK inhibitors as well as synthetic approachesfor preparing PTK inhibitors are described in the literature and in, forexample, U.S. Pat. No. 5,521,184. Each of these (and other) PTKinhibitors can be covalently attached (either directly or through one ormore atoms) to a water-soluble, non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:

R₁ is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-loweralkyl-substituted phenyl wherein the amino group in each case is free,alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom, or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen;

R₂ and R₃ are each independently of the other hydrogen or lower alkyl;

one or two of the radicals R₄, R₅, R₆, R₇ and R₈ are each nitro,fluoro-substituted lower alkoxy or a radical of Formula II-C,—N(R₉)—C(═Z)—(Y)_(n)—R₁₀—X-POLY  (Formula II-C),wherein

R₉ is hydrogen or lower alkyl,

Z is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-loweralkyl-hydroximino,

Y is oxygen or —NH—,

n is 0 or 1,

R₁₀ is a bivalent aliphatic hydrocarbon radical having 5-22 carbonatoms, a phenyl or naphthyl radical, each of which is unsubstituted orsubstituted by a moiety selected from the group consisting of cyano,lower alkyl, hydroxy-lower alkyl, amino-lower alkyl,(4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-loweralkylamino, lower alkanoylamino, benzoylamino, carboxy, loweralkoxycarbonyl, phenyl-lower alkyl wherein the phenyl radical isunsubstituted or substituted, a cycloalkyl or cycloalkenyl radicalhaving up to 30 carbon atoms, cycloalkyl-lower alkyl orcycloalkenyl-lower alkyl each having up to 30 carbon atoms in thecycloalkyl or cycloalkenyl moiety, a monocyclic radical having 5 or 6ring members and 1-3 ring hetero atoms selected from nitrogen, oxygenand sulfur, to which radical one or two benzene radicals may be fused,and lower alkyl substituted by a monocyclic radical,

X is a spacer moiety, and

POLY is a water-soluble, non-peptidic oligomer; and

the remaining radicals of R₄, R₅, R₆, R₇ and R₈ that are not nitro,fluoro-substituted lower alkoxy or a radical of formula II are eachindependently selected from the group consisting of hydrogen, loweralkyl that is unsubstituted or substituted by free or alkylated amino,piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or loweralkanoyl, trifluoromethyl, free, etherified or esterifed hydroxy, free,alkylated or acylated amino or free or esterified carboxy.

Exemplary compounds of the invention include those moieties of FormulaI-C wherein: R₁ is unsubstituted or C₁₋₇ alkyl-substituted pyridylbonded at a ring carbon atom; R₂ is selected from the group consistingof H, methyl and ethyl; R₃ is selected from the group consisting of H,methyl and ethyl; only one of R₄, R₅, R₆, R₇ and R₈ is a radical of—N(R₉)—C(═Z)—(Y)_(n)—R₁₀—X-POLY (Formula II-C), and the remaining of R₄,R₅, R₆, R₇ and R₈ are each independent selected from the group ofhydrogen, methyl and ethyl, and further wherein, with respect to FormulaII-C, R₉ is selected from the group consisting of H, methyl and ethyl, Zis oxo, (n) is 0, and R₁₀ is a bivalent aromatic radical (such as2-naphthyl and phenyl) optionally substituted with cyano, hydroxyl,amino, and 4-methyl-piperazinyl substituted methyl, X is a spacermoiety, and POLY is a water-soluble, non-peptidic oligomer.

Further exemplary compounds of the invention are

wherein:

R₂ and R₃ are each independently of the other hydrogen or lower alkyl;

R₉ is hydrogen or lower alkyl,

Y is oxygen or —NH—,

n is 0 or 1,

R₁₀ is a bivalent aliphatic hydrocarbon radical having 5-22 carbonatoms, a phenyl or naphthyl radical, each of which is unsubstituted orsubstituted by a moiety selected from the group consisting of cyano,lower alkyl, hydroxy-lower alkyl, amino-lower alkyl,(4-methyl-piperazinyl)-lower alkyl, trifluoromethyl, hydroxy, loweralkoxy, lower alkanoyloxy, halogen, amino, lower alkylamino, di-loweralkylamino, lower alkanoylamino, benzoylamino, carboxy, loweralkoxycarbonyl, phenyl-lower alkyl wherein the phenyl radical isunsubstituted or substituted, a cycloalkyl or cycloalkenyl radicalhaving up to 30 carbon atoms, cycloalkyl-lower alkyl orcycloalkenyl-lower alkyl each having up to 30 carbon atoms in thecycloalkyl or cycloalkenyl moiety, a monocyclic radical having 5 or 6ring members and 1-3 ring hetero atoms selected from nitrogen, oxygenand sulfur, to which radical one or two benzene radicals may be fused,and lower alkyl substituted by a monocyclic radical,

X is a spacer moiety, and

POLY is a water-soluble, non-peptidic oligomer; and

each of R₄, R₆, R₇ and R₈ are each independently selected from the groupconsisting of hydrogen, lower alkyl, or lower alkanoyl, trifluoromethyl,free, etherified or esterifed hydroxy, free, alkylated or acylated aminoor free or esterified carboxy.

Use of discrete oligomers (e.g., from a monodisperse or bimodalcomposition of oligomers, in contrast to relatively impure compositions)to form oligomer-containing compounds are preferred. For instance, acompound of the invention, when administered by any of a number ofsuitable administration routes, such as parenteral, oral, transdermal,buccal, pulmonary, or nasal, exhibits reduced penetration across theblood-brain barrier. It is preferred that the compounds of the inventionexhibit slowed, minimal or effectively no crossing of the blood-brainbarrier, while still crossing the gastro-intestinal (GI) walls and intothe systemic circulation if oral delivery is intended. Moreover, thecompounds of the invention maintain a degree of bioactivity as well asbioavailability in comparison to the bioactivity and bioavailability ofthe compound free of all oligomers.

With respect to the blood-brain barrier (“BBB”), this barrier restrictsthe transport of drugs from the blood to the brain. This barrierconsists of a continuous layer of unique endothelial cells joined bytight junctions. The cerebral capillaries, which comprise more than 95%of the total surface area of the BBB, represent the principal route forthe entry of most solutes and drugs into the central nervous system.

For compounds whose degree of blood-brain barrier crossing ability isnot readily known, such ability may be determined using a suitableanimal model such as an in situ rat brain perfusion (“RBP”) model asdescribed herein. Briefly, the RBP technique involves cannulation of thecarotid artery followed by perfusion with a compound solution undercontrolled conditions, followed by a wash out phase to remove compoundremaining in the vascular space. (Such analyses may be conducted, forexample, by contract research organizations such as Absorption Systems,Exton, Pa.). In one example of the RBP model, a cannula is placed in theleft carotid artery and the side branches are tied off. A physiologicbuffer containing the analyte (typically but not necessarily at a 5micromolar concentration level) is perfused at a flow rate of about 10mL/minute in a single pass perfusion experiment. After 30 seconds, theperfusion is stopped and the brain vascular contents are washed out withcompound-free buffer for an additional 30 seconds. The brain tissue isthen removed and analyzed for compound concentrations via liquidchromatography with tandem mass spectrometry detection (LC/MS/MS).Alternatively, blood-brain barrier permeability can be estimated basedupon a calculation of the compound's molecular polar surface area(“PSA”), which is defined as the sum of surface contributions of polaratoms (usually oxygens, nitrogens and attached hydrogens) in a molecule.The PSA has been shown to correlate with compound transport propertiessuch as blood-brain barrier transport. Methods for determining acompound's PSA can be found in, e.g., Ertl et al. (2000) J. Med. Chem.43:3714-3717 and Kelder (1999) Pharm. Res. 16:1514-1519.

With respect to the blood-brain barrier, the water-soluble, non-peptidicoligomer-small molecule drug conjugate exhibits a blood-brain barriercrossing rate that is reduced as compared to the crossing rate of thesmall molecule drug not attached to the water-soluble, non-peptidicoligomer. Exemplary reductions in blood-brain barrier crossing rates forthe compounds described herein include reductions of: at least about 5%;at least about 10%; at least about 25%; at least about 30%; at leastabout 40%; at least about 50%; at least about 60%; at least about 70%;at least about 80%; or at least about 90%, when compared to theblood-brain barrier crossing rate of the small molecule drug notattached to the water-soluble oligomer. A preferred reduction in theblood-brain barrier crossing rate for a conjugate of the invention is atleast about 20%.

Assays for determining whether a given compound (regardless of whetherthe compound includes a water-soluble, non-peptidic oligomer) can act asa PTK inhibitor are known and/or may be prepared by one of ordinaryskill in the art and are further described herein.

Briefly, however, an exemplary method for determining whether a givencompound (regardless of whether the compound includes a water-soluble,nonpeptidic oligomer) can inhibit the enzyme protein kinase C, involvesobtaining protein kinase C from pig brain and purifying it in accordancewith the procedure described by Uchida et al. (1984) J. Biol. Chem.259:12311-4. The protein kinase C-inhibiting activity of a givencompound can be determined following the approach described in Fabbro etal. (1985) Arch. Biochem. Biophys. 239:102-111.

Each of these (and other) PTK inhibitor moieties can be covalentlyattached (either directly or through one or more atoms) to awater-soluble, non-peptidic oligomer.

Exemplary molecular weights of a small molecule PTK inhibitor moiety(prior to including a water-soluble, non-peptidic oliogmer) includemolecular weights of: less than about 950; less than about 900; lessthan about 850; less than about 800; less than about 750; less thanabout 700; less than about 650; less than about 600; less than about550; less than about 500; less than about 450; less than about 400; lessthan about 350; and less than about 300 Daltons.

The small molecule drug used in the invention, if chiral, may beobtained from a racemic mixture, or an optically active form, forexample, a single optically active enantiomer, or any combination orratio of enantiomers (e.g., scalemic and racemic mixtures). In addition,the small molecule drug may possess one or more geometric isomers. Withrespect to geometric isomers, a composition can comprise a singlegeometric isomer or a mixture of two or more geometric isomers. A smallmolecule drug for use in the present invention can be in its customaryactive form, or may possess some degree of modification. For example, asmall molecule drug may have a targeting agent, tag, or transporterattached thereto, prior to or after covalent attachment of an oligomer.Alternatively, the small molecule drug may possess a lipophilic moietyattached thereto, such as a phospholipid (e.g.,distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolamine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

The PTK inhibitor moiety for coupling to a water-soluble, non-peptidicoligomer possesses a free hydroxyl, carboxyl, thio, amino group, or thelike (i.e., “handle”) suitable for covalent attachment to the oligomer.In addition, the PTK inhibitor moiety may be modified by introduction ofa reactive group, preferably by conversion of one of its existingfunctional groups to a functional group suitable for formation of astable covalent linkage between the oligomer and the drug.

Each oligomer is composed of up to three different monomer typesselected from the group consisting of: alkylene oxide, such as ethyleneoxide or propylene oxide; olefinic alcohol, such as vinyl alcohol,1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkyl methacrylamideor hydroxyalkyl methacrylate, where alkyl is preferably methyl;α-hydroxy acid, such as lactic acid or glycolic acid; phosphazene,oxazoline, amino acids, carbohydrates such as monosaccharides, alditolsuch as mannitol; and N-acryloylmorpholine. Preferred monomer typesinclude alkylene oxide, olefinic alcohol, hydroxyalkyl methacrylamide ormethacrylate, N-acryloylmorpholine, and α-hydroxy acid. Preferably, eacholigomer is, independently, a co-oligomer of two monomer types selectedfrom this group, or, more preferably, is a homo-oligomer of one monomertype selected from this group.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) can have any of a number of differentgeometries. For example, the water-soluble, non-peptidic oligomer can belinear, branched, or forked. Most typically, the water-soluble,non-peptidic oligomer is linear or is branched, for example, having onebranch point. Although much of the discussion herein is focused uponpoly(ethylene oxide) as an illustrative oligomer, the discussion andstructures presented herein can be readily extended to encompass anywater-soluble, non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, theoligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 monomers in series. Thus, for example, when thewater-soluble, non-peptidic polymer includes CH₃—(OCH₂CH₂)_(n)—, “n” isan integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, andcan fall within one or more of the following ranges: between about 1 andabout 25; between about 1 and about 20; between about 1 and about 15;between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to the PTKinhibitor (in contrast to the step-wise addition of one or more monomersto effectively “grow” the oligomer onto the PTK inhibitor), it ispreferred that the composition containing an activated form of thewater-soluble, non-peptidic oligomer be monodisperse. In thoseinstances, however, where a bimodal composition is employed, thecomposition will possess a bimodal distribution centering around any twoof the above numbers of monomers. For instance, a bimodal oligomer mayhave any one of the following exemplary combinations of monomersubunits: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, and so forth;2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, and so forth; 3-4, 3-5, 3-6,3-7, 3-8, 3-9, 3-10, and so forth; 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and soforth; 5-6, 5-7, 5-8, 5-9, 5-10, and so forth; 6-7, 6-8, 6-9, 6-10, andso forth; 7-8, 7-9, 7-10, and so forth; and 8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut”, to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y., Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication No.2005/0136031.

When present, the spacer moiety (through which the water-soluble,non-peptidic polymer is attached to the PTK inhibitor moiety) may be asingle bond, a single atom, such as an oxygen atom or a sulfur atom, twoatoms, or a number of atoms. A spacer moiety is typically but is notnecessarily linear in nature. The spacer moiety, “X,” is hydrolyticallystable, and is preferably also enzymatically stable. Preferably, thespacer moiety “X” is one having a chain length of less than about 12atoms, and preferably less than about 10 atoms, and even more preferablyless than about 8 atoms and even more preferably less than about 5atoms, whereby length is meant the number of atoms in a single chain,not counting substituents. For instance, a urea linkage such as this,R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to have a chain lengthof 3 atoms (—NH—C(O)—NH—). In selected embodiments, the linkage does notcomprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) other atoms,as described further below.

More specifically, in selected embodiments, a spacer moiety of theinvention, X, may be any of the following: “—” (i.e., a covalent bond,that may be stable or degradable, between the PTK inhibitor residue andthe water-soluble, non-peptidic oligomer), —O—, —NH—, —S—, —C(O)—,—C(O)O—, —OC(O)—, —CH₂—C(O)O—, —CH₂—OC(O)—, —C(O)O—CH₂—, —OC(O)—CH₂—,C(O)—NH, NH—C(O)—NH, O—C(O)—NH, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH —, —NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂,—CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—, —NH—CH₂—CH₂—,—CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl. Additionalspacer moieties include, acylamino, acyl, aryloxy, alkylene bridgecontaining between 1 and 5 inclusive carbon atoms, alkylamino,dialkylamino having about 2 to 4 inclusive carbon atoms, piperidino,pyrrolidino, N-(lower alkyl)-2-piperidyl, morpholino, 1-piperizinyl,4-(lower alkyl)-1-piperizinyl, 4-(hydroxyl-lower alkyl)-1-piperizinyl,4-(methoxy-lower alkyl)-1-piperizinyl, and guanidine. In some instances,a portion or a functional group of the drug compound may be modified orremoved altogether to facilitate attachment of the oligomer. In someinstances, it is preferred that X is not an amide, i.e., —CONR— or—RNCO—).

For purposes of the present invention, however, a group of atoms is notconsidered a linkage when it is immediately adjacent to an oligomersegment, and the group of atoms is the same as a monomer of the oligomersuch that the group would represent a mere extension of the oligomerchain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is formed by reaction of a functional group on a terminusof the oligomer (or nascent oligomer when it is desired to “grow” theoligomer onto the PTK inhibitor) with a corresponding functional groupwithin the PTK inhibitor. Illustrative reactions are described brieflybelow. For example, an amino group on an oligomer may be reacted with acarboxylic acid or an activated carboxylic acid derivative on the smallmolecule, or vice versa, to produce an amide linkage. Alternatively,reaction of an amine on an oligomer with an activated carbonate (e.g.,succinimidyl or benzotriazolyl carbonate) on the drug, or vice versa,forms a carbamate linkage. Reaction of an amine on an oligomer with anisocyanate (R—N═C═O) on a drug, or vice versa, forms a urea linkage(R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group onan oligomer with an alkyl halide, or halide group within a drug, or viceversa, forms an ether linkage. In yet another coupling approach, a smallmolecule having an aldehyde function is coupled to an oligomer aminogroup by reductive amination, resulting in formation of a secondaryamine linkage between the oligomer and the small molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.

The termini of the water-soluble, non-peptidic oligomer not bearing afunctional group may be capped to render it unreactive. When theoligomer includes a further functional group at a terminus other thanthat intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional groupcomprises an electrophilic or nucleophilic group for covalent attachmentto a small molecule, depending upon the reactive group contained withinor introduced into the small molecule. Examples of nucleophilic groupsthat may be present in either the oligomer or the small molecule includehydroxyl, amine, hydrazine (—NHNH₂), hydrazide (—C(O)NHNH₂), and thiol.Preferred nucleophiles include amine, hydrazine, hydrazide, and thiol,particularly amine. Most small molecule drugs for covalent attachment toan oligomer will possess a free hydroxyl, amino, thio, aldehyde, ketone,or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, 2-thiazolidine thione, etc., as wellas hydrates or protected derivatives of any of the above moieties (e.g.,aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative that reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbomene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g., hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.,aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the PTK inhibitor may not have a functional groupsuited for conjugation. In this instance, it is possible to modify (or“functionalize”) the “original” PTK inhibitor so that it does have afunctional group suited for conjugation. For example, if the PTKinhibitor has an amide group, but an amine group is desired, it ispossible to modify the amide group to an amine group by way of a Hofmannrearrangement, Curtius rearrangement (once the amide is converted to anazide) or Lossen rearrangement (once amide is concerted to hydroxamidefollowed by treatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of small molecule PTK inhibitorbearing a carboxyl group wherein the carboxyl group-bearing smallmolecule PTK inhibitor is coupled to an amino-terminated oligomericethylene glycol, to provide a conjugate having an amide group covalentlylinking the small molecule PTK inhibitor to the oligomer. This can beperformed, for example, by combining the carboxyl group-bearing smallmolecule PTK inhibitor with the amino-terminated oligomeric ethyleneglycol in the presence of a coupling reagent, (such asdicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a small molecule PTKinhibitor bearing a hydroxyl group wherein the hydroxyl group-bearingsmall molecule PTK inhibitor is coupled to an oligomeric ethylene glycolhalide to result in an ether (—O—) linked small molecule conjugate. Thiscan be performed, for example, by using sodium hydride to deprotonatethe hydroxyl group followed by reaction with a halide-terminatedoligomeric ethylene glycol.

Further, it is possible to prepare a conjugate of a small molecule PTKinhibitor moiety bearing a hydroxyl group wherein the hydroxylgroup-bearing small molecule PTK inhibitor moiety is coupled to anoligomeric ethylene glycol bearing an haloformate group [e.g.,CH₃(OCH₂CH₂)_(n)OC(O)-halo, where halo is chloro, bromo, iodo] to resultin a carbonate [—O—C(O)—O—] linked small molecule conjugate. This can beperformed, for example, by combining a PTK inhibitor moiety and anoligomeric ethylene glycol bearing a haloformate group in the presenceof a nucleophilic catalyst (such as 4-dimethylaminopyridine or “DMAP”)to thereby result in the corresponding carbonate-linked conjugate.

In another example, it is possible to prepare a conjugate of a smallmolecule PTK inhibitor bearing a ketone group by first reducing theketone group to form the corresponding hydroxyl group. Thereafter, thesmall molecule PTK inhibitor now bearing a hydroxyl group can be coupledas described herein.

In still another instance, it is possible to prepare a conjugate of asmall molecule PTK inhibitor bearing an amine group. In one approach,the amine group-bearing small molecule PTK inhibitor and analdehyde-bearing oligomer are dissolved in a suitable buffer after whicha suitable reducing agent (e.g., NaCNBH₃) is added. Following reduction,the result is an amine linkage formed between the amine group of theamine group-containing small molecule PTK inhibitor and the carbonylcarbon of the aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a small molecule PTKinhibitor bearing an amine group, a carboxylic acid-bearing oligomer andthe amine group-bearing small molecule PTK inhibitor are combined, inthe presence of a coupling reagent (e.g., DCC). The result is an amidelinkage formed between the amine group of the amine group-containingsmall molecule PTK inhibitor and the carbonyl of the carboxylicacid-bearing oligomer.

While it is believed that the full scope of the conjugates disclosedherein behave as described, an optimally sized oligomer can beidentified as follows.

First, an oligomer obtained from a monodisperse or bimodal water solubleoligomer is conjugated to the small molecule drug. Preferably, the drugis orally bioavailable, and on its own, exhibits a non-negligibleblood-brain barrier crossing rate. Next, the ability of the conjugate tocross the blood-brain barrier is determined using an appropriate modeland compared to that of the unmodified parent drug. If the results arefavorable, that is to say, if, for example, the rate of crossing issignificantly reduced, then the bioactivity of conjugate is furtherevaluated. Preferably, the compounds according to the invention maintaina significant degree of bioactivity relative to the parent drug, i.e.,greater than about 30% of the bioactivity of the parent drug, or evenmore preferably, greater than about 50% of the bioactivity of the parentdrug.

The above steps are repeated one or more times using oligomers of thesame monomer type but having a different number of subunits and theresults compared.

For each conjugate whose ability to cross the blood-brain barrier isreduced in comparison to the non-conjugated small molecule drug, itsoral bioavailability is then assessed. Based upon these results, that isto say, based upon the comparison of conjugates of oligomers of varyingsize to a given small molecule at a given position or location withinthe small molecule, it is possible to determine the size of the oligomermost effective in providing a conjugate having an optimal balancebetween reduction in biological membrane crossing, oral bioavailability,and bioactivity. The small size of the oligomers makes such screeningsfeasible and allows one to effectively tailor the properties of theresulting conjugate. By making small, incremental changes in oligomersize and utilizing an experimental design approach, one can effectivelyidentify a conjugate having a favorable balance of reduction inbiological membrane crossing rate, bioactivity, and oralbioavailability. In some instances, attachment of an oligomer asdescribed herein is effective to actually increase oral bioavailabilityof the drug.

For example, one of ordinary skill in the art, using routineexperimentation, can determine a best suited molecular size and linkagefor improving oral bioavailability by first preparing a series ofoligomers with different weights and functional groups and thenobtaining the necessary clearance profiles by administering theconjugates to a patient and taking periodic blood and/or urine sampling.Once a series of clearance profiles have been obtained for each testedconjugate, a suitable conjugate can be identified.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

To determine whether the PTK inhibitor or the conjugate of a PTKinhibitor and a water-soluble, non-peptidic oligomer has activity as aPTK inhibitor therapeutic, it is possible to test such a compound. ThePTK inhibitor compounds may be tested using in vitro binding studies toreceptors using various cell lines expressing these receptors that havebecome routine in pharmaceutical industry and described herein.

Enzyme Assay. The assays may be carried out using the protein tyrosinekinases Lck, Fyn, Lyn, lick, Fgr, Src, Blk and Yes.

The particular protein tyrosine kinase of interest is incubated inkinase buffer (20 mM MOPS, pH7, 10 mM MgCl₂) in the presence of the testcompound. The reaction is initiated by the addition of substrates to thefinal concentration of 1 μM ATP, 3.3 μCi/ml [³³P] gamma-ATP, and 0.1mg/ml acid denatured enolase (prepared as described in Cooper et al.(1984) J. Biol. Chem. 259:7835-7841). The reaction is stopped after tenminutes by the addition of 10% trichloroacetic acid, 100 mM sodiumpyrophosphate followed by 2 mg/ml bovine serum albumin. The labeledenolase protein substrate is precipitated at 4° C., harvested ontoPackard Unifilter plates and counted in a scintillation counter toascertain the protein tyrosine kinase inhibitory activity of the testconjugate (activity inversely proportional to the amount of labeledenolase protein obtained). The exact concentration of reagents and theamount of label can be varied as needed.

Enzyme Assay Using HER1 or HER2. Conjugates of interest are assayed in akinase buffer that containing 20 mM Tris.HCl, pH 7.5, 10 mM MnCl₂, 0.5mM dithiothreitol, bovine serum albumin at 0.1 mg/ml, poly(glu/tyr, 4:1)at 0.1 mg/ml, 1 μM ATP, and 4 μCi/ml [gamma³³P]ATP. Poly(glu/tyr, 4:1)is a synthetic polymer that serves as a phosphoryl acceptor and ispurchased from Sigma Chemicals. The kinase reaction is initiated by theaddition of enzyme and the reaction mixtures are incubated at 26° C. forone hour. The reaction is terminated by the addition of EDTA to 50 mMand proteins are precipitated by the addition of trichloroacetic acid to5%. The precipitated proteins are recovered by filtration onto PackardUnifilter plates and the amount of radioactivity incorporated ismeasured in a Topcount scintillation counter.

Cell Assays. Cellular Tyrosine Phosphorylation. Jurkat T cells areincubated with the test compound and then stimulated by the addition ofantibody to CD3 (monoclonal antibody G19-4). Cells are lysed after fourminutes or at another desired time by the addition of a lysis buffercontaining NP-40 detergent. Phosphorylation of proteins is detected byanti-phosphotyrosine immunoblotting. Detection of phosphorylation ofspecific proteins of interest such as ZAP-70 is detected byimmunoprecipitation with anti-ZAP-70 antibody followed byanti-phosphotyrosine immunoblotting. Such procedures are described inSchieven et al. (1994) Journal of Biological Chemistry 269:20718-20726and the references incorporated therein. The Lck inhibitors inhibit thetyrosine phosphorylation of cellular proteins induced by anti-CD3antibodies.

Calcium Assay. Lck inhibitors block calcium mobilization in T cellsstimulated with anti-CD3 antibodies. Cells are loaded with the calciumindicator dye, such as indo-1, treated with anti-CD3 antibody such asthe monoclonal antibody G19-4, and calcium mobilization is measuredusing flow cytometry by recording changes in the blue/violet indo-1ratio as described in Schieven et al. (supra).

Proliferation Assays: Lck inhibitors inhibit the proliferation of normalhuman peripheral blood T cells stimulated to grow with anti-CD3 plusanti-CD28 antibodies. A 96 well plate is coated with a monoclonalantibody to CD3 (such as G19-4), the antibody is allowed to bind, andthen the plate is washed. The antibody bound to the plate serves tostimulate the cells. Normal human peripheral blood T cells are added tothe wells along with test compound plus anti-CD28 antibody to provideco-stimulation. After a desired period of time (e.g., 3 days), the[³H]-thymidine is added to the cells, and after further incubation toallow incorporation of the label into newly synthesized DNA, the cellsare harvested and counted in a scintillation counter to measure cellproliferation.

The compounds of the invention may be tested in animal models of cancersto determine their cancer-inhibition potential.

Other assays include tumor regression assays in animal models, asdescribed in, for example, U.S. Pat. No. 5,521,184.

The compounds of the invention may be administered per se or in the formof a pharmaceutically acceptable salt, and any reference to thecompounds of the invention herein is intended to includepharmaceutically acceptable salts. If used, a salt of a compound asdescribed herein should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compound or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Such pharmacologically and pharmaceutically acceptable saltscan be prepared by reaction of the compound with an organic or inorganicacid, using standard methods detailed in the literature. Examples ofuseful salts include, but are not limited to, those prepared from thefollowing acids: hydrochloric, hydrobromic, sulfuric, nitric,phosphoric, maleic, acetic, salicyclic, p-toluenesulfonic, tartaric,citric, methanesulfonic, formic, malonic, succinic,naphthalene-2-sulphonic and benzenesulphonic, and the like. Also,pharmaceutically acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium, or calcium salts of acarboxylic acid group.

The present invention also includes pharmaceutical preparationscomprising a compound as provided herein in combination with apharmaceutical excipient. Generally, the compound itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, maltitol, lactitol, xylitol, sorbitol,myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines, fatty acidsand fatty esters; steroids, such as cholesterol; and chelating agents,such as EDTA, zinc and other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. The optimal amount of any individual excipient isdetermined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethylcellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form (as alyophilizate or precipitate, which can be in the form of a powder orcake), as well as formulations prepared for injection, which are liquidand require the step of reconstituting the dry form of parenteralformulation. Examples of suitable diluents for reconstituting solidcompositions prior to injection include bacteriostatic water forinjection, dextrose 5% in water, phosphate-buffered saline, Ringer'ssolution, saline, sterile water, deionized water, and combinationsthereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions,normally being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The compounds of the invention can also be administered through the skinusing conventional transdermal patch or other transdermal deliverysystem, wherein the conjugate is contained within a laminated structurethat serves as a drug delivery device to be affixed to the skin. In sucha structure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The compounds of the invention can also be formulated into a suppositoryfor rectal administration. With respect to suppositories, the compoundis mixed with a suppository base material which is (e.g., an excipientthat remains solid at room temperature but softens, melts or dissolvesat body temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the compound (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

In some embodiments of the invention, the compositions comprising thecompounds of the invention may further be incorporated into a suitabledelivery vehicle. Such delivery vehicles may provide controlled and/orcontinuous release of the compounds and may also serve as a targetingmoiety. Non-limiting examples of delivery vehicles include, adjuvants,synthetic adjuvants, microcapsules, microparticles, liposomes, and yeastcell wall particles. Yeast cells walls may be variously processed toselectively remove protein component, glucan, or mannan layers, and arereferred to as whole glucan particles (WGP), yeast beta-glucan mannanparticles (YGMP), yeast glucan particles (YGP), Rhodotorula yeast cellparticles (YCP). Yeast cells such as S. cerevisiae and Rhodotorulaspecies are preferred; however, any yeast cell may be used. These yeastcells exhibit different properties in terms of hydrodynamic volume andalso differ in the target organ where they may release their contents.The methods of manufacture and characterization of these particles aredescribed in U.S. Pat. Nos. 5,741,495, 4,810,646, 4,992,540, 5,028,703,5,607,677 and U.S. Patent Application Publication Nos. 2005/0281781 and2008/0044438.

The invention also provides a method for administering a compound of theinvention as provided herein to a patient suffering from a conditionthat is responsive to treatment with the compound. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe compound (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of a particular compound ofthe invention. Those of ordinary skill in the art appreciate whichconditions a specific compound can effectively treat. Exemplaryconditions for which the compounds of the present invention are believedto be useful include chronic myelogenous leukemia (CML),gastrointestinal stromal tumors (GISTS), renal cell carcinoma, and solidtumors, including breast, lung, and colorectal cancers. The actual doseto be administered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given compound of the invention (again,preferably provided as part of a pharmaceutical preparation) can beadministered in a variety of dosing schedules depending on the judgmentof the clinician, needs of the patient, and so forth. The specificdosing schedule will be known by those of ordinary skill in the art orcan be determined experimentally using routine methods. Exemplary dosingschedules include, without limitation, administration five times a day,four times a day, three times a day, twice daily, once daily, threetimes weekly, twice weekly, once weekly, twice monthly, once monthly,and any combination thereof. Once the clinical endpoint has beenachieved, dosing of the composition is halted.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All non-PEG chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

¹H NMR (nuclear magnetic resonance) data was generated by an NMRspectrometer. A list of certain compounds as well as the source of thecompounds is provided below.

Example 1 Synthesis of Compounds Based on Imatinib

Preparation of 3-(dimethylamino)-1-(pyridine-3-yl) prop-2-en-1-one(compound 4): Acetylpyridine (11 mL, 0.1 mol) and N,N′-dimethylformamidedimethylacetal (27 mL, 0.2 mol) were dissolved in o-xylene (35 mL). Themixture was heated to 130° C. with stirring for 20 hours. During thereaction the methanol formed was removed by a trap connected to thereflux condenser. Upon cooling to room temperature, hexane (20 ml) wasadded to the mixture. The precipitated solid was collected by suctionfiltration and washed with hexane (200 mL). The desired product wasobtained (15.4 g, yield: 90%) as a white solid; ¹H NMR (500 MHz, CDCl₃)δ b 2.95 (s, 3H), 3.18 (s, 3H), 5.68 (d, 1H), 7.36 (m, 1H), 7.84 (d,1H), 8.18 (d, 1H), 8.67 (m, 1H), 9.10 (s, 1H). LC-MS (m/z) calculated,176.1, found 177.1 [M+H]+.

Preparation of 4-(pyridine-3-yl) pyridine-2-amine (compound 5):3-(Dimethylamino)-1-(pyridine-3-yl) prop-2-en-1-one (15.4 g, 0.088 mol),guanidine nitrate (10.7 g, 0.088 mol), and sodium hydroxide (3.5 g,0.088 mol) were dissolved in n-butanol (120 mL). The mixture was heatedto reflux with stirring and maintained for 16 hours. The reactionmixture was then cooled to room temperature. The solid was collected andwashed with water (400 mL). The desired product was dried under vacuumfor three days and 12.7 g of yellow crystals of desired product wasobtained (yield: 85%); ¹H NMR (500 MHz, CDCl₃) δ 5.15 (br., 2H), 7.09(d, 1H), 7.44 (in, 1H), 8.33 (d, 1H), 8.42 (d, 1H), 8.73 (d, 1H), 9.22(s, 1H). LC-MS (m/z) calculated, 172.2, found 173.2 [M+H]+.

Preparation of N-(2-methyl-5-nitrophenyl)-4—(pyridine-3-yl)pyridine-2-amine (compound 6): 4-(Pyridine-3-yl) pyridine-2-amine (8.60g, 0.05 mol), CuI (2.39 g, 0.0125 mmol), anhydrous K₂CO₃ (14.0 g, 0.1mol), KI (2.08 g, 0.0125 mol) were added to a Schlenk-type three-neckflask fitted with a thermometer, magnetic stirbar, and septum. The flaskwas evacuated and back filled with N₂ three times. Dioxane (250 mL) and2-bromo-4-nitrotoluene (9.82 g, 0.045 mol) were added to the flask.Finally, N,N′-dimethylethylenediamine (1.10 g, 0.0125 mol) was added bysyringe at room temperature with stirring. The reaction mixture wasstirred at 110° C. under N₂ for 24 hours and then cooled to roomtemperature. Ammonia (30%, 100 mL) and saturated NaCl (400 mL), wereadded to the reaction mixture which was then extracted with ethylacetate (500 mL×3). The organic phase was dried over Na₂SO₄ and a yellowsolid was obtained after the solvent was removed under reduced pressure.The crude product was purified by column chromatography (biotage:DMC/Methanol, 1-8%, 20 CV). The desired product was obtained as a yellowsolid (8.3 g, yield: 60%); ¹H NMR (500 MHz, CDCl₃) δ 2.49 (s, 3H), 7.36(m, 1H), 7.15 (br., 1H), 7.35 (d, 1H), 7.38 (d, 1H), 7.52 (m, 1H), 7.88(d, 1H), 8.55 (d, 1H), 8.60 (d, 1H), 8.76 (d, 1H), 9.28 (s, 1H), 9.50(s, 1H). LC-MS (m/z) calculated, 307.3, found, 308.3 [M+H]+.

Preparation of 6-Methyl-N-(4-(pyridine-3-yl) pyrimidin-2-yl)benzene-1,3-diamine (compound 7):N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyridine-2-amine (3.07 g,10.0 mmol), hydrazine monohydrate (1.54 g of 65% solution in water, 20.0mmol), FeCl₃ (21 mg, 0.13 mmol), and activated carbon (0.2 g) weredissolved in a mixture of methanol (200 mL) and ethyl acetate (100 mL).The reaction mixture was heated to 80° C. with stirring and maintainedfor 24 hours. The reaction mixture was cooled own to room temperature.The insoluble materials were removed by filtration, and the filtrate wasconcentrated under reduced pressure. The resulting residue was dissolvedin ethyl acetate (150 mL) and washed with water (150×2). The organicphase was dried over anhydrous Na₂SO₄. The solvent was removed underreduced pressure and the resulting residue was purified by columnchromatography (biotage: DMC/Methanol, 1-8%, 25 CV). The product wasobtained (1.57 g, yield: 56.4%) as a yellow solid and 0.53 g startingmaterial of N-(2-methyl-5-nitrophenyl)—4-(pyridine-3-yl)pyridine-2-amine was recovered; ¹H NMR (500 MHz, CDCl₃) δ 2.28 (s, 3H),3.68 (s, 2H), 6.45 (d, 1H), 6.95 (s, 1H), 7.02 (d, 1H), 7.18 (d, 1H),7.45 (m, 1H), 7.35 (d, 1H), 7.65 (s, 1H), 8.37 (d, 1H), 8.52 (d, 1H),8.74 (d, 1H), 9.28 (s, 1H). LC-MS (m/z) calculated, 277.3, found, 278.2[M+H]+.

Preparation of 4-(Chloromethyl)-N-(4-(methyl-3-(4—(pyridin-3-yl)pyrimidine-2-ylamino)phenyl) benzamide (compound 8):6-Methyl-N-(4-(pyridine-3-yl) pyrimidin-2-yl) benzene-1,3-diamine (1.24g, 4.46 mmol) and TEA (1.4 mL, 8.92 mmol) was dissolved in THF (15 mL).The resulting solution was cooled to 0° C. with stirring and maintainedfor 10 minutes. A solution of 4-(chloromethyl)benzoyl chloride (0.97 g,5.14 mmol) in THF (5 mL) was added dropwise. After stirring at 0° C. for4 hours, water (140 mL) was added dropwise to the reaction mixture, anda light-yellow precipitate appeared. The resulting precipitate wascollected by suction filtration, washed with water (300 mL), and driedunder vacuum. The crude product was purified by column chromatography(biotage: DMC/Methanol, 1-8%, 25 CV). The desired product was obtained(1.82 g, yield: 94.2%) as a light-yellow solid; ¹H NMR (500 MHz, CDCl₃)δ 2.38 (s, 3H), 4.66 (s, 2H), 7.05 (s, 1H), 7.22 (m, 2H), 7.30 (m, 1H),7.42 (d, 1H), 7.51 (d, 2H), 7.89 (m, 3H), 8.51 (m, 2H), 8.63 (s, 1H),8.71 (d, 1H), 9.28 (s, IH). LC-MS (m/z) calculated, 429.14, found, 430.2[M+H]+.

Synthesis of mPEG_(n)-N-Imatinib:4—(Chloromethyl)-N-(4-(methyl-3-(4-(pyridin-3—yppyrimidine-2—ylamino)phenyl)benzamide(129 mg, 0.3 mmol) and mPEG_(n)-NH₂ (1.50 mmol, n=3, 5, 7, 9) wasreacted under microwave conditions at 120° C. for one hour. Aftercooling to room temperature, DCM (100 mL) was added to the reactionmixture. The resulting solution was extracted with 1M HCl (50 mL). Theacidic aqueous phase was neutralized with sodium carbonate, and thenextracted with DCM (100 mL×2). The organic phase was dried over Na₂SO₄and the solvent removed under reduced pressure. The resulting residuewas purified by column chromatography (biotage: DCM/CH₃OH, CH₃OH, 5—10%,25 CV). The desired products were obtained in yields: 65-85%.

mPEG₃-N-Imatinib (compound la) NMR (500 MHz, CDCl₃) δ 2.27 (s, 3H), 2.62(br., 1H), 2.76 (t, 2H), 3.31 (s, 3H), 3.49 (m, 2H), 3.59 (m, 8H), 3.80(s, 2H), 7.08 (s, 1H), 7.12 (m, 1H), 7.28 (m, IH), 7.30 (m, 3H), 7.79(d, 2H), 8.42 (m, 2H), 8.51(d, 2H), 8.62 (d, 1H), 9.17 (s, 1H); LC-MS(m/z) calculated, 556.3, found, 557.3 [M+H]+.

mPEG₅-N-Imatinib (compound 1b) ¹H NMR (500 MHz, CDCl₃) δ 1.99 (br., 1H),2.35 (s, 3H), 2.80 (t, 2H), 3.34 (s, 3H), 3.52 (m, 2H), 3.62 (m, 16H),3.86 (s, 2H), 7.06 (s, 1H), 7.17 (m, 2H), 7.30 (m, 1H), 7.41 (m, 3H),7.84 (d, 2H), 8.11 (s, 1H), 8.51 (m, 2H), 8.59 (s, 1H), 8.69 (d, 1H),9.22 (s, 1H); LC-MS (m/z) calculated, 644.3, found, 645.3 [M +H]+.

mPEG₇-N-Imatinib (compound 1c)¹H NMR (500 MHz, CDCl₃) δ 1.93 (br., 1H),2.33 (s, 3H), 2.80 (t, 2H), 3.34 (s, 3H), 3.52 (m, 2H), 3.62 (m, 24H),3.86 (s, 2H), 7.06 (s, 1H), 7.17 (m, 2H), 7.30 (m, 1H), 7.41 (m, 3H),7.84 (d, 2H), 8.11 (s, 1H), 8.51(m, 2H), 8.59 (s, 1H), 8.69 (d, 1H),9.22 (s, 1H); LC-MS (m/z) calculated, 732.4, found, 733.5 [M +H]+.

mPEG₉-N-Imatinib (compound 1d) NMR (500 MHz, CDCl₃)δ 2.27 (s, 3H), 276(t, 2H), 3.31 (s, 3H), 3.48 (m, 2H), 3.58 (m, 32H), 3.81 (s, 2H), 7.10(m, 3H), 7.28 (m, 1H), 7.37 (m, 3H), 7.84 (d, 2H), 8.44 (m, 2H), 8.46(m, 1H), 8.52 (m, 2H), 8.63 (d, 1H), 9.17 (s, 1H); LC-MS (m/z)calculated, 820.4, found, 821.5 [M+H]+.

Preparation of mPEG_(n)-N-piperazine: t-Butyl 1-piperazine carboxylate(372 mg, 2.0 mmol) and mPEG_(n)—Br (2.40 mmol, n=3, 5, 7, and 9) weredissolved in DMF (1 mL) and potassium carbonate (414 mg, 3.0 mmol) inwater (0.5 mL) was added. The reaction was carried out with a CEMmicrowave system at 100° C. for one hour. The reaction mixture wastransferred to a separatory funnel with DCM (100 mL), and washed withwater (100 mL×2). The organic phase was dried over Na₂SO₄ and thesolvent removed under reduced pressure. The oil obtained was dissolvedin 5 mL of a TFAIDCM mixture (2:3) which was stirred for three hours atroom temperature. Water (15 mL) was added to the reaction mixture andthe acidic aqueous phase was washed with DCM (30 mL×2). The aqueousphase was neutralized with sodium carbonate, and then extracted with DCM(100 mL×2). The combined organic phase was dried over anhydrousNa₂SO₄and the solvent removed under reduced pressure. The desiredproducts were obtained as colorless oils which were used in the nextreaction without further purification.

mPEG₃-N-piperazine ¹H NMR (500 MHz, CDCl₃) δ 2.49 (br., 4H), 2.61 (t,2H), 2.94 (t, 4H), 3.38 (s, 3H), 3.55 (m, 2H), 3.64 (m, 8H).

mPEG₅-N-piperazine NMR (500 MHz, CDCl₃) δ 2.50 (br., 4H), 2.61 (t, 2H),2.94 (t, 4H), 3.36 (s, 3H), 3.55 (m, 2H), 3.64 (m, 16H).

mPEG₇-N-piperazine ¹H NMR (500 MHz, CDCl₃) δ 2.48 (br., 4H), 2.61 (t,2H), 2.94 (t, 4H), 3.38 (s, 3H), 3.55 (m, 2H), 3.65 (m, 24H).

mPEG₉-N-piperazine ¹H NMR (500 MHz, CDCl₃) δ 2.53 (br., 4H), 2.60 (t,2H), 2.96 (t, 4H), 3.38 (s, 3H), 3.55 (m, 2H), 3.64 (m, 32H).

Preparation of mPEG_(n)-N′-Imatinib:4-(Chloromethyl)-N-(4-(methyl-3-(4-(pyridin-3-yl)pyrimidine-2-ylamino)phenyl)benzamide(189 mg, 0.44 mmol) and mPEG_(n)-piperazine (0.44 mmol, n=3, 5, 7, 9)were dissolved in DMF (1 mL) and potassium carbonate (91 mg, 0.66 mmol)in water (0.5 mL) was added. The reaction was carried out with a CEMmicrowave system at 100° C. for one hour. Upon cooling to roomtemperature, DCM (100 mL) was added to the reaction mixture and theresulting solution was washed with water (100 mL×2). The organic phasewas dried over Na₂SO₄ and the solvent removed under reduced pressure.The resulting residue was purified by column chromatography (biotage:DCM/CH₃OH, CH₃OH, 8-10%, 25 CV). The desired products were obtained inyields: 65-85%.

mPEG₃-N′-Imatinib (compound 2a) ¹H NMR (500 MHz, CDCl₃) δ 2.28 (s, 3H),2.46 (br., 8H), 2.58 (d, 2H), 3.33 (s, 3H), 3.50 (m, 4H), 3.60 (m, 8H),7.11 (m, 2H), 7.15 (m, 1H), 7.29 (m, 1H), 7.36 (m, 3H), 7.80 (d, 2H),8.29 (s, 1H), 8.43 (m, 2H), 8.54 (s, 1H), 8.64 (d, 1H), 9.18 (s, 1H);LC-MS (m/z) calculated, 625.3, found, 626.3 [M+H]+.

mPEG₅-N′-Imatinib (compound 2b)¹H NMR (500 MHz, CDCl₃) δ 2.30 (s, 3H),2.46 (br., 8H), 2.58 (d, 2H), 3.33 (s, 3H), 3.50 (m, 4H), 3.61 (m, 16H),7.11 (m, 2H), 7.15 (m, 1H), 7.29 (m, 1H), 7.36 (m, 3H), 7.80 (d, 2H),8.29 (s, 1H), 8.43 (m, 2H), 8.54 (s, 1H), 8.64 (d, 1H), 9.18 (s, 1H);LC-MS (in z) calculated, 713.4, found, 714.5 [M+H]+.

mPEG₇-N′-Imatinib (compound 2c) ¹H NMR (500 MHz, CDCl₃) δ 2.32 (s, 3H),2.48 (br., 8H), 2.58 (d, 2H), 3.35 (s, 3H), 3.54 (m, 4H), 3.62 (m, 24H),7.06 (s, 1H), 7.16 (m, 2H), 7.19 (m, 1H), 7.29 (m, 1H), 7.41 (m, 3H),7.80 (d, 2H), 8.10 (s, 1H), 8.43 (m, 2H), 8.54 (s, 1H), 8.64 (d, 1H),9.21 (s, 1H); LC-MS (m/z) calculated, 801.4, found, 802.5 [M+H]+.

mPEG₉-N′-Imatinib (compound 2c) ¹H NMR (500 MHz, CDCl₃) δ 2.34 (s, 3H),2.57 (br., 4H), 2.73 (br., 611), 3.36 (s, 3H), 3.53 (in, 2H), 3.62 (m,34H), 7.09 (s, 1H), 7.17 (m, 2H), 7.34 (in, 1H), 7.41 (m, 3H), 7.85 (d,2H), 8.19 (br., 1H), 8.51 (n, 3H), 8.71 (d, 1H), 9.30 (s, 1H); LC-MS(m/z) calculated, 889.5, found, 890.5 [M+H]+.

Example 2 ABL and PDGFR, and C-kit Tyrosine Kinase Inhibition

These assays were completed using the Caliper LABCHIP 3000 and a12-sipper LABCHIP. LABCHIP assays are separations-based, meaning thatthe product and substrate are electrophoretically separated, therebyminimizing interferences and yielding the highest data quality availableon any screening platform. Z′ factors for both the EZ Reader and LC3000enzymatic assays are routinely in the 0.8 to 0.9 range. High Z′ values,few false positives, few false negatives and analytical qualityreproducibility are the reasons cited for the increasing reliance on theLABCI-IIP assays.

The off-chip incubation mobility-shift kinase assay uses a microfluidicchip to measure the conversion of a fluorescent peptide substrate to aphosphorylated product. The reaction mixture, from a microtiter platewell, is introduced through a capillary sipper onto the chip, where thenon-phosphorylated substrate and phosphorylated product are separated byelectrophoresis and detected via laser-induced fluorescence. Thesignature of the fluorescence signal over time reveals the extent of thereaction. The assay conditions are provided in Table 1. DMSO and DTTwere at 4% and 1 mM, respectively.

TABLE 1 Assay Conditions Kinase Final [E] Peptide ATP (at Km)Buffer/Detergent Cation Enzyme Vendor/Cat # ABL 0.7 nM 1.5 μM  14 μM 50mM HEPES, pH MgCl₂ MILLIPORE/ FL-labeled 7.5/0.002% BRIJ 14-529 PDGFRβ 50 nM 1.5 μM  12 μM 50 mM MOPS, pH MnCl₂ MILLIPORE/ FL-labeled6.5/0.004% TRITON 14-463 X-100 cKIT  20 nM 1.5 μM 300 μM 50 mM Hepes, pH7.5 MnCl₂ MILLIPORE/ FL-labeled 14-559

Results shown are the averages of replicate wells. A result of >3E-06 isreported for curves that did not reach 50% activity at the highestconcentration chosen for the study. Activity must be ≦50% to report anaccurate IC₅₀. ABL AVG % activity at a specific concentration isprovided in Table 2, PDGFR AVG % activity at specific concentration isprovided in Table 3, and C-kit AVG % activity at specific concentrationis provided in Table 4. Imatinib mesylate was obtained from commercialsources and all other compounds were obtained in accordance with theprocedures set forth in Example 1.

TABLE 2 ABL AVG % Activity at Specific Concentration Compound 3.0E−061.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09 1.0E−09 3.0E−10 1.0E−10IC₅₀ (M) mPEG₃-N-Imatinib 49 61 71 78 85 92 96 96 97 97 1.2E−06mPEG₅-N-Imatinib 66 74 80 86 92 95 96 97 96 96   3E−06 mPEG₇-N-Imatinib59 73 82 90 94 94 96 97 97 97   3E−06 mPEG₉-N-Imatinib 70 77 85 89 93 9796 96 96 96   3E−06 mPEG₃-N′-Imatinib 43 66 79 84 89 92 94 98 97 962.7E−06 mPEG₅-N′-Imatinib 38 61 76 82 87 92 98 96 97 99 1.9E−06mPEG₇-N′-Imatinib 52 69 78 83 88 92 96 96 97 96   3E−06mPEG₉-N′-Imatinib 49 69 79 84 89 93 96 97 97 97 3.8E−06 Imatinibmesylate 2 11 33 59 74 79 86 91 94 97 1.6E−07

TABLE 3 PDGFR AVG % Activity at Specific Concentration Compound 3.0E−061.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09 1.0E−09 3.0E−10 1.0E−10IC₅₀ (M) mPEG₃-N-Imatinib 0 4 19 43 67 89 100 90 96 97 8.2E−08mPEG₅-N-Imatinib 0 9 37 62 85 94 100 90 92 92 2.2E−07 mPEG₇-N-Imatinib 013 39 67 100 96 99 93 93 97 2.3E−07 mPEG₉-N-Imatinib 0 11 38 65 75 83100 78 86 89 2.9E−07 mPEG₃-N′-Imatinib 0 12 35 53 69 79 81 89 100 933.9E−07 mPEG₅-N′-Imatinib 0 9 31 50 80 79 98 93 95 100 1.5E−07mPEG₇-N′-Imatinib 0 18 43 76 91 91 93 98 100 95 3.1E−07mPEG₉-N′-Imatinib 0 13 48 74 86 95 100 99 95 92 3.4E−07 Imatinibmesylate 0 4 15 36 73 87 94 98 100 99 6.9E−08

TABLE 4 C-kit AVG % Activity at Specific Concentration Compound 3.0E−051.0E−05 3.0E−06 1.0E−06 3.0E−07 1.0E−07 3.0E−08 1.0E−08 3.0E−09 1.0E−09IC₅₀ (M) mPEG₃-N-Imatinib 0 1 4 15 47 69 88 91 97 NA  2.4E−07mPEG₅-N-Imatinib 0 1 9 28 59 82 91 92 96 NA  4.4E−07 mPEG₇-N-Imatinib 16 23 50 76 88 95 100 99 NA  1.0E−06 mPEG₉-N-Imatinib 2 9 32 62 81 93 9998 99 NA  1.6E−06 mPEG₃-N′-Imatinib 0 1 9 30 60 80 91 95 96 NA  4.5E−07mPEG₅-N′-Imatinib 0 3 13 37 66 84 89 87 91 NA 7.60E−07 mPEG₇-N′-Imatinib4 16 44 69 84 91 97 95 95 NA  2.5E−06 mPEG₉-N′-Imatinib 0 2 12 35 64 8292 91 95 NA  5.5E−07 Imatinib-NMe 1 0 3 13 42 69 87 94 97 95  2.2E−07Imatinib mesylate 3 13 40 69 86 93 96 97 NA NA  2.1E−07

Example 3 Thermodynamic Solubility of Imatinib-Corresponding Compounds

Test compound (2.5 mg of solid; n=1) is weighed in a clear glass vialand buffer (0.5 mL) is added (phosphate buffered saline, pH 7.4). Thesolution is agitated at ambient temperature overnight using a vialroller system. The solution is then filtered (0.45 AM pore size; withoutpre-saturation). Duplicate aliquots (50 μL) are sampled from thefiltrate and diluted with one volume of 0.1 N hydrochloric acid andmethanol (1:1 v/v) before analysis by HPLC-UV. A standard is prepared inDMSO at 10 mg/mL (n=1) which is then diluted 10 fold in 0.1 Nhydrochloric acid and methanol (1:1 v/v) to give a 1 mg/mL solution. Theconcentration of test compound in the filtrate is quantified relative tothe concentration standard.

Analysis is performed using a gradient HPLC-UV system with a total cycletime of six minutes. The UV detection is performed using a photodiodcarray detector acquired between 220 nm and 300 nm and total response ismonitored. The results are shown in Table 5, below. Imatinib wasobtained from commercial sources and all other compounds were obtainedin accordance with the procedures set forth in Example 1

TABLE 5 Solubility Results for Tested Compounds Solubility Compound(mg/mL) Imatinib 0.06 mPEG₃-N′-imatinib 0.5 mPEG₅-N′-imatinib 3.3mPEG₇-N′-imatinib 3.0 mPEG₉-N′-imatinib 2.5 Imatinib-NMe 2.0mPEG₃-N-imatinib 3.3 mPEG₅-N-imatinib 3.4 mPEG₇-N-imatinib 3.6mPEG₉-N-imatinib 2.7

Example 4 Caco-2 Permeability (Bi-directional; pH 7.4/pH 7.4)

Caco-2 cells are used as an in vitro model of the human intestinalepithelium and permit assessment of the intestinal permeability ofpotential drugs. Test compound is added to either the apical orbasolateral side of a confluent monolayer of Caco-2 cells andpermeability is measured by monitoring the appearance of the testcompound on the opposite side of the membrane using LC-MS/MS. Apparentpermeability (P_(app)) coefficients, efflux ratio for the test compoundand recovery values were determined.

To measure the permeability of test compound in the apical tobasolateral (A-B) and basolateral to apical (B-A) direction acrossCaco-2 cells. A ratio of B-A and A-B permeabilities is calculated(efflux ratio) which shows whether the compound undergoes activetransport.

Caco-2 cells obtained from the ATCC are used between passage numbers40-60. Cells are seeded on to Millipore Multiscreen Caco-2 plates at1×10⁵ cells/cm². They are cultured for 20 days in DMEM and media ischanged every two or three days. On day 20 the permeability study isperformed.

Hanks Balanced Salt Solution (HBSS) pH 7.4 buffer with 25 mM HEPES and4.45 mM glucose at 37° C. is used as the medium in the permeabilitystudies. Incubations are carried out in an atmosphere of 5% CO₂ with arelative humidity of 95% at 37° C. On day 20, the monolayers areprepared by rinsing both basolateral and apical surfaces twice with HBSSat 37° C. Cells are then incubated with HBSS in both apical andbasolateral compartments for 40 min to stabilize physiologicalparameters. HBSS is then removed from the apical compartment andreplaced with test compound dosing solutions. The solutions are made bydiluting 10 mM test compound in DMSO with HBSS to give a final testcompound concentration of 10 μM (final DMSO concentration 1%). Thefluorescent integrity marker lucifer yellow is also included in thedosine solution. Analytical standards are made from dosing solutions.The apical compartment inserts are then placed into ‘companion’ platescontaining fresh HBSS. For basolateral to apical (B-A) permeabilitydetermination the experiment is initiated by replacing buffer in theinserts then placing them in companion plates containing dosingsolutions. At 120 minutes the companion plate is removed and apical andbasolateral samples diluted for analysis by LC-MS/MS. Test compoundpermeability was assessed in duplicate. On each plate, compounds ofknown permeability characteristics were run as controls.

Test and control compounds were quantified by LC-MS/MS cassette analysisusing a 5-point calibration with appropriate dilution of the samples.Cyprotex generic analytical conditions were used. The startingconcentration (C₀) and experimental recovery were calculated from bothapical and basolateral compartment concentrations.

The integrity of the monolayers throughout the experiment was checked bymonitoring lucifer yellow permeation using fluorimetric analysis.Lucifer yellow permeation is low if monolayers have not been damaged. Ifa lucifer yellow P_(app) value was above QC limits in one individualtest compound well, then an n=1 result is reported. If lucifer yellowP_(app) values are above QC limits in both replicate wells for a testcompound, the compound was re-tested. If on repeat, high lucifer yellowpermeation was observed in both wells then toxicity or inherentfluorescence of the test compound was assumed and no further experimentswere performed.

Data Analysis: The permeability coefficient for each compound (P_(app))is calculated from the following equation:

⁢P app = ( ⅆ Q ⅆ t )wherein dQ/dt is the rate of permeation of the drug across the cells, C₀is the donor compartment concentration at time zero and A is the area ofthe cell monolayer. C₀ is obtained from analysis of donor and receivercompartments at the end of the incubation period. It is assumed that allof the test compound measured after 120 minutes incubation was initiallypresent in the donor compartment at 0 minutes. An efflux ratio (ER) isderived as follows:

${ER} = \frac{P_{{app}{({B - A})}}}{P_{{app}{({A - B})}}}$

An efflux ratio greater than two shows efflux from the Caco-2 cells,which indicates that the compound may have potential absorption problemsin vivo (although in such an instance, other delivery approaches can beuseful to address potential absorption problems).

The apparent permeability (P_(app (A-B))) values of test compounds werecompared to those of control compounds, atenolol and propranolol, whichhave human absorption of approximately 50 and 90% respectively.Talinolol (a known P-gp substrate) is also included as a controlcompound to assess whether functional P-gp is present in the Caco-2 cellmonolayer. The apparent permeability and efflux ratio of testedcompounds are shown in Table 6, below. Imatinib was obtained fromcommercial sources and all other compounds were obtained in accordancewith the procedures set forth in Example 1

TABLE 6 Apparent Permeability and Efflux Ratios of Tested CompoundsCompound Permeability (cm²/s × 10⁶) Efflux ratio Imatinib 9.0 ± 0.5 2.89mPEG₃-N′-imatinib 4.0 ± 0.8 8.15 mPEG₅-N′-imatinib 1.0 +0.2 38.2mPEG₇-N′-imatinib  0.3 ± 0.01 75.7 mPEG₉-N′-imatinib 0.05 ± 0.02 359Imatinib-NMe 1.2 ± 0.4 60.4 mPEG₃-N-imatinib  0.6 ± 0.07 79.1mPEG₅-N-imatinib 0.05 ± 0.02 560 mPEG₇-N-imatinib 0.04 ± 0.01 391mPEG₉-N-imatinib  0.01 ± 0.004 482

Example 5 Tumor-Inhibiting Activity

The tumor-inhibiting activity is determined using female Balb/c nudemice in which human T24 bladder carcinoma has been transplanted. On day0, about 25 mg piece of solid tumor is transplanted subcutaneously underperoral “forene” narcosis on the left flank and the small incision woundis closed with a suture clip. On day 6 after the tumor transplantation,the mice are randomized in groups of 6 animals and treatment iscommenced. The treatment is carded out for 15 days by administering acompound of the invention or the corresponding compound without awater-soluble, non-peptidic oligomer in different doses perorally orintraperitoneally once daily. The tumors are measured twice weekly witha sliding caliper and the tumor volume is determined. In this assay, theadministration of a compound of the invention effects a reduction in theaverage tumor volume compared with the corresponding compound without awater-soluble, non-peptidic oligomer.

What is claimed is:
 1. A method of treating a condition selected fromthe group consisting of chronic myelogenous leukemia, gastrointestinalstromal tumors, renal cell carcinoma, breast cancer, lung cancer andcolorectal cancer, the method comprising administering to a patient inneed thereof a compound selected from

and pharmaceutically acceptable salts thereof, wherein X is a spacermoiety selected from a covalent bond, —C(O), —C(O)O—, —CH₂C(O)O—,CH₂—OC(O)—, —C(O)O—CH₂—, —C(O)—NH, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂, —CH₂—O—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O —, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂,—CH₂—NH—CH₂, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—CH₂—,—CH₂—CH₂—C(O)—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, a bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl; and POLY is apoly(alkylene oxide) oligomer.
 2. The method of claim 1, wherein thepoly(alkylene oxide) oligomer is a poly(ethylene oxide) oligomer.
 3. Themethod of claim 2, wherein the poly(ethylene oxide) oligomer has fromabout 1 to about 30 monomers.
 4. The method of claim 3, wherein thepoly(ethylene oxide) oligomer has from about 1 to about 10 monomers. 5.The method of claim 2, wherein the poly(alkylene oxide) includes analkoxy or hydroxy end-capping moiety.
 6. The method of claim 1, whereinX is selected from a covalent bond, —C(O), —C(O)O—, —CH₂C(O)O—,—CH₂—OC(O)—, —C(O)O—CH₂—, —C(O)—NH, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂ , —CH₂—O—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂, —C(O)—CH₂—, —C(O)—CH₂—CH₂—,and —CH₂—CH₂—C(O)—.
 7. The method of claim 1, wherein the X is selectedfrom a covalent bond, —C(O), —C(O)O—, —C(O)—NH—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, and —CH₂—CH₂—CH₂—CH₂.
 8. The method of claim 1, wherein Xis a covalent bond.
 9. The method of claim 1, wherein the compound isselected from

wherein n is an integer from 1 to
 30. 10. The method of claim 9, whereinn is an integer from 2 to 10.